L-Aspartate beta-decarboxylase gene (Asd) from Pseudomonas dacunhae was heterologously expressed in Escherichia coli. However, it showed low activity under acidic condition, thereby limiting its wide application in industrial process. In this study, we aimed to improve the activity of Asd under acidic condition by using site mutation. The non-conservative residues located in the substrate entrance tunnel were selected, and the enzymatic properties of variants were investigated. Finally, the whole-cell transformation was performed with variants. The activities of most variants were significantly lower than those of wild type (WT, 65.95 U/mg), except N34D, which showed higher activity of 71.67 U/mg. The mutant N34D showed higher activity at the pH range from 3.5 to 5.5, and the relative activity was over 82% at pH 5.0, which was considerably higher than that of the wild-type (50%). The activity of mutant N34D at pH range of 5.0-8.5 was the same with the wild-type. Under the optimal catalytic conditions, the production of L-alanine by N34D was 1.5 times higher than that of the wild-type. The activity of variant N34D was successfully increased under acidic condition, which shows a promising application for the production of L-alanine at an industrial scale.